Cationic oligospermine-oligonucleotide conjugates provide carrier-free splice switching in monolayer cells and in spheroids,

We report the evaluation of 18-mer 2’-O-methyl-modified ribose oligonucleotides with a full-length phosphorothioate backbone chemically conjugated at the 5’-end to the oligospermine units [Sn-: n = 5, 15, 20, 25, 30 (number of spermine units)] as splice switching oligonucleotides (SSOs). These conjugates contain, in their structure, covalently linked oligocation moieties, making them capable of penetrating cells without transfection vector. In cell culture, we observed efficient cytoplasmic and nuclear delivery of fluorescein labelled S20-SSO by fluorescent microscopy. The SSO conjugates containing more than 15 spermine units induced significant carrier-free exon-skipping at nanomolar concentration in the absence and in the presence of serum. With an increasing number of spermine units, the conjugates became slightly toxic but more active. Advantages of these molecules were particularly demonstrated in 3D cell culture (MCTS: multicellular tumor spheroids) that mimics living tissues. While vector-complexed SSOs displayed a drastically reduced splice switching in MCTS compared to the assay in monolayer culture, an efficient exon-skipping without significant toxicity was observed with oligospermine grafted SSOs (S15- and S20-SSOs) transfected without vector. It was shown, by flow cytometry and confocal microscopy, that the fluorescein-labelled S20-SSO was freely diffusing and penetrating the innermost cells of MCTS while the vector-complexed SSO penetrated only the cells of the spheroid's outer layer.